The plasma membrane calcium ATPase 2 (PMCA2) transports calcium into milk and regulates mammary epithelial cell (MEC) death during post-lactation involution. Furthermore, PMCA2 expression in breast cancers correlates inversely with patient survival. NHERF1 is a scaffolding molecule that utilizes PDZ- motif interactions to connect ion channels, ion pumps and receptors to the actin cytoskeleton in order to facilitate signaling complexes at the apical surface of epithelial cells. We now have generated considerable preliminary data showing that PMCA2 interacts with NHERF1 and ErbB2/HER2/Neu (HER2) to regulate MEC survival and HER2 signaling during lactation. Preliminary data also suggest that genetic manipulation of PMCA2 expression profoundly affects HER2-mediated tumor formation. Our overarching hypothesis is that PMCA2, NHERF1 and HER2 form a functional complex during lactation that is required to transport calcium out of MECs, to maintain active HER2 signaling and to prevent premature MEC death. We propose that, after weaning, distension of the MECs in response to milk stasis disrupts this complex, which causes an increase in intracellular calcium and an inhibition of HER2 signaling, both of which combine to activate the lysosomal permeability (LMP) pathway of MEC death. Finally, we propose that the PMCA2/NHERF1/HER2 complex is re-expressed and activates HER2 signaling during malignant transformation, supporting the survival of emerging cancer cells and driving the progression of HER2-positive breast cancers. In order to test this hypothesis, we propose 4 specific aims. Aim 1 will examine whether PMCA2, NHERF1 and HER2 interact within a multi-protein complex during lactation and whether milk stasis during early involution disrupts this complex. Aim 2 will examine whether PMCA2 regulates a calcium-calpain-LMP pathway of cell death and HER2 activity during lactation and involution. Aim 3 will examine whether loss of NHERF1 mimics loss of PMCA2 by activating the LMP pathway and inhibiting HER2 activity during involution. Aim 4 will examine whether PMCA2 and/or NHERF1 regulate HER2-mediated tumor formation in mice. These studies will advance our knowledge of the LMP pathway of cell death, will help us better understand how milk stasis initiates mammary involution and will provide new insight into the regulation of HER2 expression, localization and signaling during lactation and during malignant transformation. Finally, our studies will validate novel pathway for potential new therapeutic targets in HER2-positive breast cancer.